The iron fist of the immune system

Lcn2, center, binds to bacterial enterochelin, depriving it of iron essential for bacterial growth. The illustration in the upper right shows a macrophage cell engulfing bacteria. Chemical structures are depicted for the E. coli iron-binding molecules aerobactin, left, and enterochelin, right.

Illustration Courtesy of Dr. Roland Strong

A new study has found that when it comes to battling certain bacterial infections, the immune system fights back with an iron fist.

In a paper published online in Nature, Dr. Roland Strong and colleagues demonstrate that an immune-system protein called lipocalin 2 (Lcn2) protects mice from infection with intestinal bacteria, particularly E. coli, by depriving the microbes of iron. Lcn2 — recently renamed siderocalin — works by sequestering a bacterial compound that scavenges trace amounts of iron, an element essential for the growth of all organisms.

Since human cells also produce Lcn2, the researchers speculate that it could be developed into a new kind of antibiotic to boost an individual's innate infection-fighting ability. Because many other microbes extract iron from their surroundings in a manner similar to that used by E. coli, Lcn2 potentially could be used to combat a variety of bacterial pathogens.

Potential therapy

"The therapeutic potential for this protein is treatment of drug-resistant or otherwise intractable bacterial infections, or to treat patients who have low counts of infection-fighting cells or are otherwise immunocompromised as a result of chemotherapy or transplant, for instance," said Strong, an investigator in the Basic Sciences Division. "We're currently looking for other related proteins that are specific for different types of bacteria, and we are also engineering advantageous binding properties into Lcn2 itself."

The study was led by Dr. Alan Aderem, Dr. Trude Flo, and colleagues at the Institute for Systems Biology in Seattle. Dr. Shizuo Akira's laboratory at the Research Institute for Microbial Diseases in Osaka, Japan, was also involved. Dr. Meg Holmes, a staff scientist in Strong's lab at the time the work was done, was a co-author.

The findings described in the paper illustrate the power of open communication in science, said Strong, whose laboratory specializes in the use of x-ray crystallography to decipher the three-dimensional structure of proteins. In 2002, his group published a paper on the structure of Lcn2 (known then as NGAL) that also provided evidence that Lcn 2 might be used by the immune system to fight bacteria. His group showed that Lcn2 binds to a bacterial iron-binding molecule called enterochelin, which scavenges iron from the microbe's environment. What Strong's lab lacked, though, were the tools to test whether this binding was important for the immune response in a living animal.

That opportunity arose after Strong began speaking about his group's ideas for the function of the protein in public seminars, and several local collaborators — including Aderem — learned of the work. Aderem's lab has conducted extensive research on immune-system cells, called macrophages, which produce Lcn2. Coincidentally, a postdoc in Aderem's lab who had previously worked with Akira remembered that Akira's group had generated a strain of mice that was unable to produce Lcn2.

"This strain of mice had shown no obvious defects at the time it was developed," Strong said. "But no one had tested whether the inability to produce Lcn2 affected their ability to mount an immune response against a bacterial infection."

Lcn2 response

Previous studies had demonstrated that Lcn2 is one of many proteins that macrophages produce in response to bacterial infection. Macrophages help to protect the body from infection by ingesting invading microbes. Although Lcn2 levels become elevated during infection, researchers had not determined its precise role in immune function.

In the current study, the researchers found that levels of Lcn2 in the blood rose about 30-fold when normal mice were infected with E. coli. Next, they examined the growth of E. coli in blood serum extracted from normal mice infected with the bacteria and compared it to bacterial growth in serum extracted from Lcn2-deficient mice after infection. Bacterial levels were 1,000 times higher in serum from the Lcn2-deficient mice than in serum from normal infected mice, suggesting that the protein was responsible for the growth inhibition. Most significant was the finding that a dose of E.coli that is survivable by 100 percent of normal mice kills more than 80 percent of Lcn2-deficient animals.

The researchers found that the Lcn2's antibacterial activity only worked against bacteria that depend exclusively on the iron-scavenging molecule enterochelin. Although all E. coli strains produce enterochelin, some also produce additional iron-scavenging molecules, which allow them to grow and cause disease even in the presence of immune cells that make Lcn2. Enterochelin-like compounds are also produced by most other disease-causing microbes, including bugs that cause typhoid fever, diptheria and anthrax. It is not yet known whether Lcn 2 inhibits the growth of these pathogens.

Strong has recently found that Lcn2 can bind another iron-scavenging compound called carboxymycobactin, which is produced by Mycobacterium tuberculosis, the bacteria that causes tuberculosis.

His lab is currently testing a wide range of bacterial iron-scavenging compounds to see whether any bind to Lcn2 and therefore, might be susceptible to the protein's antibacterial activity.